Microscopic Colitis Patients Possess a Perturbed and Inflammatory Gut Microbiota.

BACKGROUND: Microscopic colitis (MC), an inflammatory disease of the colon, is characterized by chronic non-bloody diarrhea with characteristic inflammation and for some, collagen deposits in mucosal biopsies. The etiology of MC is unclear, although previous findings implicate luminal factors and thus the gut microbiome. However, the relationships between fecal microbiota and MC are relatively unexplored. METHODS: Stool microbiota of MC (n = 15) and healthy controls (HC; n = 21) were assessed by 16S rRNA V4 amplicon sequencing and analysis performed in QIIME. Gut microbiota functions were predicted using Piphillin and inflammatory potential assessed using an in vitro HT29 colonocyte cell assay. RESULTS: MC patient fecal microbiota were less diverse (Faiths index; p < 0.01) and compositionally distinct (PERMANOVA, weighted UniFrac, R2 = 0.08, p = 0.02) compared with HC subjects. MC microbiota were significantly depleted of members of the Clostridiales, enriched for Prevotella and more likely to be dominated by this genus (Chi2 = 0.03). Predicted pathways enriched in MC microbiota included those related to biosynthesis of antimicrobials, and sphingolipids, to glycan degradation, host defense evasion, and Th17 cell differentiation and activation. In vitro, exposure of cultured colonocytes to cell-free products of MC patient feces indicates reduced gene expression of IL-1B and occludin and increased GPR119 and the lymphocyte chemoattractant CCL20. CONCLUSION: MC gut microbiota are distinct from HC and characterized by lower bacterial diversity and Prevotella enrichment and distinct predicted functional pathways. Limited in vitro experiments indicate that compared with cell-free products from healthy fecal microbiota, MC microbiota induce distinct responses when co-cultured with epithelial cells, implicating microbiota perturbation in MC-associated mucosal dysfunction.

Three cases of Sutterella wadsworthensis bacteremia secondary to abdominal infections.

The anaerobic bacterium Sutterella wadsworthensis has previously been isolated from the human intestine, both in healthy individuals and patients with gastrointestinal disorders, and the clinical significance of this bacterium is unknown. In this case report, we describe three cases of bacteremia with Sutterella wadsworthensis, from patients with recent intraabdominal surgery.

Clinical results and microbiota changes after faecal microbiota transplantation for chronic pouchitis: a pilot study.

Objectives: Research evidence suggests that chronic pouchitis is associated with intestinal dysbiosis. Faecal microbiota transplantation (FMT) has been proposed as a possible treatment. We performed a 6-month prospective, open-label, single-centre cohort pilot-study (NCT03538366) to investigate if FMT could improve clinical outcome and alter gut microbiota in patients with chronic pouchitis.Materials and methods: Nine adult patients with chronic pouchitis were included and allocated to 14 days FMT by enemas from five faecal donors, with a 6-month follow-up. Pouchitis severity was assessed using pouchitis disease activity index (PDAI) before and after FMT. Changes in gut microbiota, and engraftment of donor's microbiota were assessed in faecal samples.Results: All patients were treated with FMT for 14 continuous days. Overall, four of nine patients receiving FMT were in clinical remission at 30-day follow-up, and three patients remained in remission until 6-month follow-up. Clinical symptoms of pouchitis improved significantly between inclusion and 14-day follow-up (p = .02), but there was no improvement in PDAI between inclusion (mean 8.6) and 30-day follow-up (mean 5.2). Treatment with FMT caused a substantial shift in microbiota and increased microbial diversity in six patients, resembling that of the donors, with a high engraftment of specific donor microbiota.Conclusions: Symptomatic benefit in FMT treatment was found for four of nine patients with chronic pouchitis with increased microbial diversity and high engraftment of donor's microbiota. A larger, randomised controlled study is required to fully evaluate the potential role of FMT in treating chronic pouchitis.

Consumption of yoghurt favours remission after faecal microbiota transplantation for chronic pouchitis.

PURPOSE: The gut microbiota is conceivably a key factor in the aetiology of pouchitis. Faecal microbiota transplantation (FMT) has been suggested as a promising new treatment for chronic pouchitis, where treatment options often are few. However, little is known about the influence of the diet on the clinical effects of FMT. We assessed the diet of patients with chronic pouchitis undergoing FMT to investigate the influence of diet on the clinical outcome after FMT. METHODS: Nine patients with chronic pouchitis were allocated to treatment with FMT delivered by enema from five faecal donors for 14 consecutive days in a 6-month prospective, open-label, single-centre cohort pilot study. A dietary questionnaire was completed at baseline for all patients and donors. Patients underwent a pouchoscopy at baseline and at 30-day follow-up, and the Pouchitis Disease Activity Index (PDAI) was assessed. RESULTS: Patients' diets were generally similar, when comparing patients in remission post-FMT (PDAI < 7) with those who relapsed (PDAI ≥ 7). Consumption of grains trended to be different between the two groups (p = 0.06), where patients in relapse consumed more bread products than did patients in remission. However, consumption of yoghurt was significantly different between the two groups (p = 0.04), with patients in remission consuming more yoghurt (mean 1.1 s/d vs 0.2 s/d). CONCLUSION: Gastroenterologist performing clinical studies on FMT for chronic pouchitis should be aware of dietary habits as contributing factors for the clinical effect of FMT.

The effect of non-pooled multi-donor faecal microbiota transplantation for inducing clinical remission in patients with chronic pouchitis: Results from a multicentre randomised double-blinded placebo-controlled trial (MicroPouch).

BACKGROUND AND AIMS: To investigate if treatment with non-pooled multi-donor faecal microbiota transplantation (FMT) for four weeks was superior to placebo to induce clinical remission in patients with chronic pouchitis. METHODS: The study was a randomised double-blinded placebo-controlled study with a 4-week intervention period and 12-month follow-up. Eligible patients with chronic pouchitis were recruited from five Danish hospitals. Participants were randomised to non-pooled multi-donor FMT derived from four faecal donors, or placebo. Treatment was delivered daily by enema for two weeks followed by every second day for two weeks. Disease severity was accessed at inclusion and 30-day follow-up, using the Pouchitis Disease Activity Index (PDAI); PDAI <7 was considered equivalent to clinical remission. Faecal samples from participants and donors were analysed by shotgun metagenomic sequencing. RESULTS: Inclusion was stopped after inclusion of 30 participants who were randomised 1:1 for treatment with FMT or placebo. There was no difference in participants achieving clinical remission between the two groups at 30-day follow-up, relative risk 1.0 (95%CI(0.55;1.81)). Treatment with FMT resulted in a clinically relevant increase in adverse events compared to placebo, incidence rate ratio 1.67 (95%CI(1.10;2.52)); no serious adverse events within either group. Faecal microbiota transplantation statistically significantly increased the similarity of participant faecal microbiome to the faecal donor microbiome at 30-days follow-up (p=0.01), which was not seen after placebo. CONCLUSIONS: Non-pooled multi-donor FMT was comparable to placebo in inducing clinical remission in patients with chronic pouchitis but showed a clinically relevant increase in adverse events compared to placebo.

Vertebral osteomyelitis caused by Campylobacter jejuni in an immunocompetent patient.

BACKGROUND: Campylobacter jejuni is the leading cause of human bacterial gastroenteritis worldwide. However, systemic infection with C. jejuni is uncommon, and osteomyelitis caused by C. jejuni is extremely rare. Cultivation from spinal bone biopsies has not previously been reported in the literature. CASE PRESENTATION: A 79-year-old immunocompetent male was admitted to the emergency department at Aalborg University Hospital in Denmark with lower back pain, fever and diarrhoea. A FecalSwab obtained upon admission was PCR-positive for Campylobacter spp, while an aerobic blood culture bottle was positive for C. jejuni (Time to detection: 70.4 h). A MRI of columna totalis showed osteomyelitis at L1/L2 with an epidural abscess from L1 to L2 with compression of the dura sack. The patient underwent spinal surgery with spondylodesis and decompression of L1/L2. The surgery was uncomplicated and the discus material was also culture positive for C. jejuni. The patient was treated with meropenem for a total duration of four weeks, followed by four weeks of oral treatment with clindamycin in tapered dosage. The patient recovered quickly following surgery and targeted antibiotic treatment with decreasing lumbar pain and biochemical response and was fully recovered at follow-up three months after end of treatment. CONCLUSIONS: While C. jejuni osteomyelitis is rare, it should still be suspected as a possible causative bacterial aetiology in patients with vertebral osteomyelitis, in particular when symptoms of diarrhoea is involved in the clinical presentation. Susceptibility testing is crucial due to emerging resistance, and targeted treatment strategies should rely upon such tests.

Fever of Unknown Origin: A Validation Study of Danish ICD-10 Diagnosis Codes.

Background: Real-world data in form of routinely collected clinical data are a valuable resource for epidemiological research in infectious disease. We examined the validity of a discharge diagnosis of fever of unknown origin from hospital discharge registries. Methods: We identified patients with a first in- or outpatient diagnosis (primary or secondary) of fever of unknown origin (ICD-10 code R50.0; R50.8, R50.9) recorded in the Danish National Patient Registry (DNPR) between 2010 and 2017 in the North Denmark Region. We based the validation cohort on a mix of patients diagnosed at a highly specialized university department of infectious diseases (n=100), other internal medicine departments (n=50), and patients diagnosed at a regional non-university hospital (n=50). We estimate positive predictive value (PPV) of diagnosis for fever of unknown origin using medical records as reference. Results: The PPV of a diagnosis of fever of unknown origin for patients diagnosed at the infectious disease department was 61% (95% CI: 51-71%). For other internal medicine departments, it was 14% (95% CI: 6-27%), and for the non-university hospital it was 16% (95% CI: 7-29%). To achieve higher PPVs, we excluded immunocompromised patients, patients who were diagnosed with infection, cancer or rheumatic disease within 7 days after admission, and/or patients with a short hospital stay (maximum 3 days) and no subsequent hospital contact within 1 month. The PPV for diagnoses from the Department of Infectious Diseases improved to 82% (95% CI: 68-91%) for other internal medicine departments it improved to 31% (95% CI: 11-59%), and for the non-university hospital it improved to 36% (95% CI: 13-65%). Conclusion: We found that only diagnoses made in the Department of Infectious Diseases accurately identified fever of unknown origin, whereas diagnoses made in other units mainly covered infection-related fever, cancer-related fever, or short unspecific fever without further diagnostic work-up.

High genetic diversity in Campylobacter concisus isolates from patients with microscopic colitis.

The emerging intestinal pathogen Campylobacter concisus has been associated with prolonged diarrhoea and classic inflammatory bowel diseases (IBD) and was recently also linked with microscopic colitis (MC). Previous reports have observed a high genetic diversity within isolates from diarrhoeic and IBD patients and from healthy controls (HC), and division of isolates into two major genomospecies (GS1 and GS2). The aim of this study was to describe genetic diversity in 80 recently cultivated MC biopsy and faecal isolates of C. concisus by multi-locus sequence typing (MLST); and to compare the phylogenetic relatedness to 102 isolates from diarrhoeic and IBD patients and HCs by k-mer-based distance estimation. MLST revealed high genetic diversity in MC isolates with 72 novel sequence types. K-mer divided MC isolates into two distinct clusters (cluster 1 n = 21, cluster 2 n = 49), with a significantly higher prevalence of cluster 2 isolates in biopsies than in faeces, p = 0.009. K-mer divided the 182 isolates into two major phylogenetic clusters: cluster 1 (GS1 isolates) and cluster 2 (GS2 isolates), which further differentiated into three subgroups. Cluster 1 and the three cluster 2 subgroups were each distinctive in mean genome size and GC count. Isolates from all disease phenotypes were present in cluster 1 and cluster 2 subgroup 2 and 3, whereas cluster 2 subgroup 1 only contained isolates restricted to patients with ulcerative colitis (n = 10) and HC (n = 4).

The Prevalence and Clinical Implications of Rectal SARS-CoV-2 Shedding in Danish COVID-19 Patients and the General Population.

Background: SARS-CoV-2 has resulted in a global pandemic since its outbreak in Wuhan, 2019. Virus transmission primarily occurs through close contact, respiratory droplets, and aerosol particles. However, since SARS-CoV-2 has been detected in fecal and rectal samples from infected individuals, the fecal-oral route has been suggested as another potential route of transmission. This study aimed to investigate the prevalence and clinical implications of rectal SARS-CoV-2 shedding in Danish COVID-19 patients. Methods: Hospitalized and non-hospitalized adults and children who were recently tested with a pharyngeal COVID-19 test, were included in the study. A rectal swab was collected from all participants. Hospitalized adults and COVID-19 positive children were followed with both pharyngeal and rectal swabs until two consecutive negative results were obtained. RT-qPCR targeting the envelope gene was used to detect SARS-CoV-2 in the samples. Demographic, medical, and biochemical information was obtained through questionnaires and medical records. Results: Twenty-eight of 52 (53.8%) COVID-19 positive adults and children were positive for SARS-CoV-2 in rectal swabs. Seven of the rectal positive participants were followed for more than 6 days. Two of these (28.6%) continued to test positive in their rectal swabs for up to 29 days after the pharyngeal swabs had turned negative. Hospitalized rectal positive and rectal negative adults were comparable regarding demographic, medical, and biochemical information. Furthermore, no difference was observed in the severity of the disease among the two groups. Conclusions: We provided evidence of rectal SARS-CoV-2 shedding in Danish COVID-19 patients. The clinical importance of rectal SARS-CoV-2 shedding appears to be minimal.

The Microbiota Profile in Inflamed and Non-Inflamed Ileal Pouch-Anal Anastomosis.

The objective was to determine the bacterial composition in inflamed and non-inflamed pouches for comparison to the microbiota of healthy individuals. Pouch patients and healthy individuals were included between November 2017 and June 2019 at the Department of Gastrointestinal Surgery, Aalborg University Hospital, Denmark. A faecal sample was collected from all participants for microbiota analysis using 16S rRNA amplicon sequencing. Overall, 38 participants were included in the study. Eleven patients with a normally functioning pouch, 9 patients with chronic pouchitis, 6 patients with familial adenomatous polyposis, and 12 healthy individuals. Patients with chronic pouchitis had overall lower microbial diversity and richness compared to patients with a normal pouch function (p < 0.001 and p = 0.009) and healthy individuals (p < 0.001 and p < 0.001). No significant difference was found between patients with familial adenomatous polyposis and chronic pouchitis (microbial diversity p = 0.39 and richness p = 0.78). Several taxa from the family Enterobacteriaceae, especially genus Escherichia, were associated primarily with patients with chronic pouchitis, while taxa from the genus Bacteroides primarily were associated with healthy individuals and patients with a normally functioning pouch. Finally, a microbial composition gradient could be established from healthy individuals through patients with normal pouch function and familial adenomatous polyposis to patients with chronic pouchitis.

Motility and biofilm formation of the emerging gastrointestinal pathogen Campylobacter concisus differs under microaerophilic and anaerobic environments.

Campylobacter concisus has been isolated from patients with gastroenteritis and inflammatory bowel disease (IBD), as well as healthy subjects. While strain differences may plausibly explain virulence differentials, an alternative hypothesis posits that the pathogenic potential of this species may depend on altered ecosystem conditions in the inflamed gut. One potential difference is oxygen availability, which is frequently increased under conditions of inflammation and is known to regulate bacterial virulence. Hence, we hypothesized that oxygen influences C. concisus physiology. We therefore characterized the effect of microaerophilic or anaerobic environments on C. concisus motility and biofilm formation, two important determinants of host colonization and dissemination. C. concisus isolates (n = 46) sourced from saliva, gut mucosal biopsies and feces of patients with IBD (n = 23), gastroenteritis (n = 8) and healthy subjects (n = 13), were used for this study. Capacity to form biofilms was determined using crystal violet assay, while assessment of dispersion through soft agar permitted motility to be assessed. No association existed between GI disease and either motility or biofilm forming capacity. Oral isolates exhibited significantly greater capacity for biofilm formation compared to fecal isolates (p<0.03), and showed a strong negative correlation between motility and biofilm formation (r = -0.7; p = 0.01). Motility significantly increased when strains were cultured under microaerophilic compared to anaerobic conditions (p<0.001). Increased biofilm formation under microaerophillic conditions was also observed for a subset of isolates. Hence, differences in oxygen availability appear to influence key physiological aspects of the opportunistic gastrointestinal pathogen C. concisus.

Molecular epidemiology and comparative genomics of Campylobacter concisus strains from saliva, faeces and gut mucosal biopsies in inflammatory bowel disease.

Campylobacter concisus is an emerging pathogen associated with inflammatory bowel disease (IBD), yet little is known about the genetic diversity of C. concisus in relation to host niches and disease. We isolated 104 C. concisus isolates from saliva, mucosal biopsies and faecal samples from 41 individuals (26 IBD, 3 Gastroenteritis (GE), 12 Healthy controls (HC)). Whole genomes were sequenced and the dataset pan-genome examined, and genomic information was used for typing using multi-locus-sequence typing (MLST). C. concisus isolates clustered into two main groups/genomospecies (GS) with 71 distinct sequence types (STs) represented. Sampling site (p < 0.001), rather than disease phenotype (p = 1.00) was associated with particular GS. We identified 97 candidate genes associated with increase or decrease in prevalence during the anatomical descent from the oral cavity to mucosal biopsies to faeces. Genes related to cell wall/membrane biogenesis were more common in oral isolates, whereas genes involved in cell transport, metabolism and secretory pathways were more prevalent in enteric isolates. Furthermore, there was no correlation between individual genetic diversity and clinical phenotype. This study confirms the genetic heterogeneity of C. concisus and provides evidence that genomic variation is related to the source of isolation, but not clinical phenotype.

Motility of Campylobacter concisus isolated from saliva, feces, and gut mucosal biopsies.

Campylobacter concisus is an emerging pathogen associated with gastrointestinal disorders such as gastroenteritis and inflammatory bowel diseases (IBD), but the species is also found in healthy subjects. The heterogeneous genome of C. concisus increases the likelihood of varying virulence between strains. Flagella motility is a crucial virulence factor for the well-recognized Campylobacter jejuni; therefore, this study aimed to analyze the motility of C. concisus isolated from saliva, gut biopsies, and feces of patients with IBD, gastroenteritis, and healthy subjects. The motility zones of 63 isolates from 52 patients were measured after microaerobic growth in soft-agar plates for 72 hours. The motility of C. concisus was significantly lower than that of Campylobacter jejuni and Campylobacter fetus subsp. fetus. The motility of C. concisus varied between isolates (4-22 mm), but there was no statistical significant difference between isolates from IBD patients and healthy subjects (p = 0.14). A tendency of a larger motility zones was observed for IBD gut mucosa isolates, although it did not reach statistical significance (p = 0.13), and no difference was found between oral or fecal isolates between groups. In conclusion, the varying motility of C. concisus could not be related to disease outcome or colonization sites.

Optimized cultivation of Campylobacter concisus from gut mucosal biopsies in inflammatory bowel disease.

BACKGROUND: Campylobacter concisus is a commensal of the human oral flora that has been linked to prolonged diarrhea and inflammatory bowel disease (IBD). It has been detected more often from intestinal biopsies in patients with IBD compared to healthy controls using PCR-based techniques, whereas the number of C. concisus culture-positive biopsies in previous studies has been very limited. Determining the rate of viable isolates present in the gut mucosa is of great importance when evaluating the role in different disease presentations. We therefore investigated a novel two-step cultivation procedure combining anaerobic and microaerobic incubation from several gut mucosal sites to improve isolate yield, and compared this to PCR results, from IBD patients and healthy controls. RESULTS: Cultivation with the novel two-step procedure yielded a higher rate of C. concisus isolates from mucosal biopsies than previously reported by other methods. From 52 IBD patients, 52/245 (21 %) biopsies were culture positive for C. concisus, while 121/245 (49 %) of biopsies were PCR positive. For 26 healthy controls, the numbers were 23/182 (13 %) and 66/182 (36 %), respectively (p < 0.001). The rate of cultivation and PCR detection was higher for IBD patients compared to healthy controls (p = 0.021, p = 0.008, respectively). CONCLUSIONS: Patients with IBD had a higher prevalence of C. concisus than healthy controls, by both cultivation and PCR detection. We found a higher rate of C. concisus isolates from gut mucosal biopsies in both IBD patients and healthy controls than in preceding studies, indicating that colonization of C. concisus in the gastrointestinal tract is more extensive than previously assumed.

Azithromycin vs. Placebo for the Clinical Outcome in Campylobacter concisus Diarrhoea in Adults: A Randomized, Double-Blinded, Placebo-Controlled Clinical Trial.

Campylobacter concisus has been associated with prolonged mild diarrhoea, but investigations regarding the efficacy of antimicrobial treatment have not been reported previously. We initiated a phase 3, single-centre, randomized, double-blinded, placebo-controlled study comparing the efficacy of 500 mg once-daily dose of azithromycin with a 500 mg once-daily dose of placebo for three days, for the treatment of C. concisus diarrhoea in adult patients with a follow-up period of ten days. If symptoms persisted at day ten, the patient was offered cross-over study treatment of three days and another ten-day follow-up period. The primary efficacy endpoint was the clinical response, defined as time to cessation of diarrhoea (<3 stools/day or reversal of accompanying symptoms). Our estimated sample size was 100 patients. We investigated a total of 10,036 diarrheic stool samples from 7,089 adult patients. Five-hundred and eighty-eight C. concisus positive patients were assessed for eligibility, of which 559 were excluded prior to randomization. The three main reasons for exclusion were duration of diarrhoea longer than 21 days (n = 124), previous antibiotic treatment (n = 113), and co-pathogens in stools (n = 87). Therefore, 24 patients completed the trial with either azithromycin (n = 12) or placebo (n = 12). Both groups presented symptoms of mild, prolonged diarrhoea with a mean duration of 18 days (95% CI: 16-19). One person in the azithromycin group and four from the placebo group chose to continue with crossover medication after the initial ten-day period. In the azithromycin group, there was a mean of seven days (95% CI: 5-9) to clinical cure and for the placebo group it was ten days (95% CI: 6-14) (OR-3 (95% CI: -7-1). We observed no differences in all examined outcomes between azithromycin treatment and placebo. However, due to unforeseen recruitment difficulties we did not reach our estimated sample size of 100 patients and statistical power to conclude on an effect of azithromycin treatment was not obtained. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT01531218.

The susceptibility of Campylobacter concisus to the bactericidal effects of normal human serum.

Campylobacter concisus is an emerging pathogen of the gastrointestinal tract that has been associated with Barrett's oesophagus, enteritis and inflammatory bowel disease. Despite having invasive potential in intestinal epithelial cells in-vitro, bacteraemic cases with C. concisus are extremely scarce, having only been reported once. Therefore, we conducted a serum resistance assay to investigate the bactericidal effects of human complement on C. concisus in comparison to some other Campylobacter species. In total, 22 Campylobacter strains were tested by incubation with normal human serum and subsequent cultivation in microaerobic conditions for 48 hours. Killing time was evaluated by decrease in total CFU over time for incubation with different serum concentrations. Faecal isolates of C. concisus showed inoculum reduction to less than 50% after 30 min. Campylobacter jejuni was sensitive to serum, but killing was delayed and a bacteraemic Campylobacter fetus subsp. fetus isolate was completely serum resistant. Interestingly, sensitivity of enteric C. concisus to human serum was not associated to different faecal-calprotectin levels. We find that faecal isolates of C. concisus are sensitive to the bactericidal effects of serum, which may explain why C. concisus is not associated to bacteraemia.

[Development of mupirocin resistance during eradication treatment of methicillin resistant Staphylococcus aureus carriage]. / Udvikling af mupirocinresistens under eradikationsbehandling ved MRSA-bærertilstand.

We report a case of a 55 year-old male who was transferred to a Danish hospital from Central America. A routine screening revealed a methicillin resistant Staphylococcus aureus (MRSA) isolate sensitive to mupirocin (minimal inhibitory concentration (MIC) 0.094 mg/L) and eradication treatment was initiated. Three weeks later the patient developed MRSA bacteraemia with the same strain, though now resistant to mupirocin (MIC 8 mg/l). The incidence of mupirocin resistance is low in Denmark compared to other countries with a heavier burden of MRSA, but there is reason to be concerned due to the increasing reliance on this drug for treatment of MRSA carriage.